The general aim of this research is to delineate the various steps in the glycosylation of proteins and to determine possible biological consequences of impaired glycosylation. Our approach is to isolate and characterize mammalian cell mutants defective in glycosylation. Chinese hamster ovary (CHO) cell mutants are isolated by both single and multistep selection procedures and by screening procedures. Once isolated, the enzymatic defect in the cell line is determined, allowing us to catalog various reactions in the synthetic pathway; subsequently, we can determine what effect the alteration in a particular reaction has on different glycoprotein products. For example, we previously determined that the defect in B4-2-1, a CHO cell line isolated by Dr. April R. Robbins, NIH, was the lack of mannosylphosphoryldolichol synthase activity. This cell line had been isolated on the basis of its altered mannose 6-phosphate receptor activity; this receptor is a glycoprotein. Recently, we have also shown that B4-2-1 lacks mannosylphosphorylretinol synthase activity, strongly implying that there is only one synthase that utilizes either dolichyl or retinyl phosphate as substrate. (A)